Detection and Characterization of a Novel Copper‐Dependent Intermediate in a Lytic Polysaccharide Monooxygenase

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper‐containing enzymes capable of oxidizing crystalline cellulose which have large practical application in the process of refining biomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report a long‐lived intermediate (t1/2=6–8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO‐CuI reacts with sub‐equimolar amounts of H2O2. UV/Vis absorption spectroscopy, electron paramagnetic resonance, resonance Raman and stopped‐flow spectroscopy suggest that the observed long‐lived intermediate involves the copper center and a nearby tyrosine (Tyr175). Additionally, activity assays in the presence of sub‐equimolar amounts of H2O2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly, this suggests that the long‐lived copper‐dependent intermediate could be part of the catalytic mechanism for LPMOs. The observed intermediate offers a new perspective into the oxidative reaction mechanism of TaLPMO9A and hence for the biomass oxidation and the reactivity of copper in biological systems.

The Role of the Active Site Tyrosine in the Mechanism of Lytic Polysaccharide Monooxygenase

“…Moreover, the reversibility of the two reactions are different: in case of TaLPMO9A the process is reversible while in case of LsAA9 the process is irreversible. 34,35 Finally, the two studies suggest different roles in the mechanism of the observed intermediates.…”

“…Yet, until very recently, this tyrosine has not been proposed to be directly involved in the mechanism [19][20][21][22] . However, two recent experimental studies have (in absence of substrate) managed to trap and spectroscopically characterize an intermediate, presumably involving a deprotonated tyrosine 34,35 . Both studies obtain well-defined optical spectra by adding H2O2 and an electron donor (ascorbate) to an AA9 LPMO.…”

“…The two studies were performed on LPMOs from Thermoascus aurantiacus (TaLPMO9A) 34 and Lentinus similis LPMO (LsAA9) 35 , respectively. Since the LPMOs belong to the same family (AA9), it was somewhat surprising that the two studies proposed different spin-states and obtain remarkably different optical spectra, suggesting that the observed intermediates are different.…”

Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase

“…To assess whether the higher photostability of the WSCP-Chl a would lead to higher TtAA9 product formation, light-driven activity assays were performed with TtAA9 using varying concentrations of reductant. Since high concentrations of H 2 O 2 can be detrimental to LPMO activity 18,40 , and the WSCP was shown to be more stable at lower light intensities (Fig. 1b), the light intensity was reduced to 100 µmol m − 2 s − 1 for TtAA9 experiments.…”

“…The cosubstrates interact with a reduced copper active site forming a reactive intermediate which can then oxidize the substrate. Recent studies have demonstrated signi cantly higher product yields when H 2 O 2 is involved in LPMO catalysis 18,19 . However, the amount of H 2 O 2 has to be controlled as high concentrations have been shown to be detrimental to LPMO activity 17 .…”